Abstract: Objective：To explore the application of internal transcribed spacer(ITS) gene sequencing in identification of pathogenic fungi. Methods：PCR amplification and gene sequencing were performed by the universal primer ITS1 and ITS4 of fungal ITS gene for 89 strains of fungi, including 6 standard strains of yeasts, 7 strains of external quality assessment, 42 strains of clinically isolated yeast like fungi and 34 strains of filamentous fungi. The amplified sequences were analyzed and compared with those of CBS database(http://www.cbs.knaw.nl/Collections) and the taxonomic information providing by Mycobank(http://cn.mycobank.org/) to obtain the information of species identification. Results：All of the 89 strains of fungi were successfully amplified into ITS target gene and the results of sequencing demonstrated the entire sequences of 89 strains were obtained. The sequence alignment showed that 5 of 6 standard strains of the yeasts were identified to species and 1 strain was identified to genus. Among 7 strains of external quality assessment, 1 strain of yeast was identified to species, 2 strains of 6 filamentous fungi were identified to species, 1 strain was identified to genus and 3 strains were identified to complex group. Among 42 strains of clinically isolated yeast like fungi, 39 were identified to species and 3 were identified to genus. Among 34 clinically isolated strains of filamentous fungi, 10 were identified to species, 18 were identified to genus and 6 were identified to complex group. Conclusion：The universal primer ITS1 and ITS4 showed broad spectrum for amplification. The identification based on amplification and sequencing analysis of ITS gene could fast, effectively identify clinical suspicious fungus, but the process of data analysis and the result interpretation should still be difficult.